PROJECT SUMMARY/ABSTRACT Inhibiting inflammation of the arterial wall by targeting interleukin-1? lowers the incidence of cardiovascular events. However, we have not discovered strategies limiting inflammation-related injury in acute ischemia. One major obstacle is the critical gap of knowledge in understanding danger recognition, the actual process that dictates the scope of inflammation. My long-term goal is to develop immune modulators that modify danger recognition to contain inflammation-mediated injury after MI. The overall objective of this proposal is to determine how DNA and its cytosolic receptor the cyclic GAMP synthase (cGAS) propagate injury triggered by ischemia. The damaged myocardium is enriched with mitochondrial (thousands of copies per cardiomyocyte) and nuclear DNA. The large amount of DNA poses a serious threat to myocardial repair when macrophages, the professional phagocytes, detect it and respond with robust inflammatory responses that are originally intended to get rid of pathogens (from the evolutionary standpoint). The central hypothesis is that recognition of DNA by cGAS sustains the inflammatory macrophages via activation of the type I interferon (IFN) pathway that promotes inflammasone activation; as a result, cGAS is crucial in ischemia-induced remodeling. This hypothesis has been formulated on the preliminary data and the recently published work from my laboratory. The rationale is that understanding the intracellular danger recognition in ischemic-triggered inflammation has the potential to discover effective ways of limiting inflammation-related injury. Guided by strong preliminary data, this hypothesis will be tested by pursuing the following specific aims: 1) Determine whether cGAS activation in macrophages drives ischemia- induced remodeling and define the source of the cytosolic DNA; 2) Determine whether cGAS sustains inflammation in macrophages by promoting AIM2 and NLRP3 inflammasome and caspase 11-mediated pyroptosis; 3) Identify effective and clinically relevant approaches for inhibition of cGAS. Aim 1 will be addressed using a cGASf/f mouse line to determine macrophage as the responsible cell type. Studies are also designed to trace the source of the cytosolic DNA. Under the second aim, I will determine if guanylate-binding proteins (GBP), induced by cGAS activation, increase danger signal visibility to sensors like AIM2 and NLRP3 and if cGAS-depndent priming is essential in cGAS-triggered inflammasome activation in ischemia. Pyroptosis- mediated by caspase 11 will also be evaluated. Aim 3 will identify clinically relevant strategies to inhibit cGAS by assessing two agents that treat inflammatory disorders. The study is conceptually novel by targeting DNA and its receptor cGAS, a bona fide anti-viral response, in the setting of myocardial ischemia. Knowledge acquired will vertically advance our understanding of the critical role of intracellular immunity in ischemic injury. As ischemic heart disease is an enormous burden and often a devastating condition, the proposed study moves the field forward by finding novel strategies alleviating the burden and improve care. Additionally, results will help to understand potential cardiac side effects from immunotherapy via boosting cGAS-STING pathway activity (in clinical trials).